The invention refers to a method for the analysis of a sample with regard to a substance contained therein.
The analysis of a liquid sample is generally concerned with the determination of the concentration of a substance (analyte) contained therein (quantitative analysis). In some cases, it is sufficient simply to determine whether the analyte is present (in a concentration exceeding a threshold value) in the sample or not (qualitative analysis). In medical applications for which the present invention is of particular importance, the analysis of body fluids (primarily blood, blood serum and urine) with regard to the analytes contained therein, such as hormones, antibodies, antigens or drugs, plays an important role.
The invention refers to the improvement of a certain type of analytic procedure which may be designated as an electrochemiluminescence binding reaction analysis (subsequently referred to as ECL-BBA standing for electrochemiluminescence biochemical binding analysis). Such a method has the following characteristic features.
a) The analytic selectivity is based on a specific biochemical binding reaction using biochemical substances which selectively can only bind to each other. Primary examples are immunological chemical binding reactions between antibodies and antigens or haptens with which the antibodies bind specifically. Other biochemical binding reactions are protein binding, in particular between avidine and biotin, the lectine carbohydrate binding, binding between receptors and ligands and the hybridization of nucleic acids.
Such specific biochemical binding reactions have been used for some time for analytic purposes. There are a plurality of differing one or multi-step reaction processes (test protocols) which finally lead, through the participation of the analyte and at least one specifically binding substance contained in the reagent system (binding reagents), to the formation of a complex characteristic for the analysis. This complex normally (but not necessarily) contains the analyte.
b) In order to render the complex, whose concentration constitutes a measure of the analytic result sought, detectable, a marking substances (label) is normally used which is coupled to a binding reagent of the reagent system, e.g. an antibody. The species comprising the marking substance and the binding reagent is designated as a conjugate.
The invention refers to cases in which the marking substance is capable of effecting an ECL-reaction. When such a substance is subjected to a suitable electrical potential on a voltametric electrode, it emits light which can be measured photometrically. A second electrochemically active substance, designated as a precursor, normally contributes to this reaction. In practice, primarily a ruthenium complex (ruthenium-tris [bipyridyl]) is used as ECL-label in combination with TPA (tripropylamine) as precursor. The two electrochemically active substances react on the electrode each releasing an electron and thereby forming a strongly reducing or oxidizing species. The subsequent redox reaction brings the ECL-label into an excited state from which it returns to the ground state with the emission of a photon. The ECL-label reaction is preferably a circular reaction so that a single label molecule emits a plurality of photons after application of a voltage to the electrode.
c) In the methods to which the invention refers, the ECL-marked complex molecules characteristic for the analysis are fixed to magnetic microparticles (beads). In practice, magnetized polystyrol balls having a diameter of typically 2 to 3 xcexcm are used. Fixing is effected by means of a pair of specific biochemical binding partners. The pair streptavidin biotin has turned out to be particularly advantageous. The beads are coated with a streptavidin polymer. Biotin is bound to the complex molecule.
The beads with the bound marked complex are introduced into the measuring cell of a measuring apparatus. The cell is equipped the electrodes (normally a working electrode, a counter electrode and, in particular for the case of a potentiometric measurement scheme, a reference electrode) which are, necessary for generating the electrical field required for triggering the ECL-reaction. The beads are drawn onto the surface of the working electrode in the magnetic field of a magnet disposed below the working electrode. Since this normally occurs in flow-through cells with continuously flowing sample fluids, the magnetic deposition of the beads is designated as xe2x80x9ccapturingxe2x80x9d.
Generally after the capturing step a washing step is carried out during which a washing fluid flows by the working electrode to remove unwanted components. An electric potential required for triggering the ECL-reaction is then applied to the working electrode and the resulting luminescence light is measured using a suitable optical detector. The intensity of the luminescence light is a measure for the concentration of the marked beads on the surface of the working electrode which, in turn, is a measure of the concentration of the analyte in the sample. A calibration allows calculation of the sought concentration from the measured luminescence signal.
A plurality of different variations of this type of ECL-BBA-method have been discussed and described in the literature. Such variations may refer to each of the individual aspects mentioned.
With regard to aspect a), the tests are distinguished from each other by different test protocols (for example sandwich tests and competitive tests, each with a plurality of different sub-variations). A fundamental difference obtains between homogeneous tests which do not require separation between the formed complex molecules and the non-complexed conjugate and heterogeneous tests which require such a bound/free separation. The present invention can be used for very differing test protocols as long as they include a reaction sequence which comprises at least one specific chemical binding reaction and which leads to the formation of a complex which is characteristic of the analysis and which is marked with an ECL-label.
Also with regard to aspect b) the invention is universally applicable, i.e. it is independent of the ECL-label used and possible additional components of the ECL-reaction. The invention has turned out to be particularly usefully for test methods using the mentioned ruthenium complex in combination with TPA.
With regard to aspect c), the invention is solely directed to tests in which the complex characteristic for the analysis is bound to magnetic microparticles and in which these microparticles are deposited on the surface of a working electrode in the magnetic field of the magnet. The invention is otherwise independent of variations of aspect c) and can e.g. be used with differing bead materials and sizes as well as differing methods for fixing the complex to the beads.
More detailed information concerning the ECL-BBA-method can be taken from the extensive literature. Towards this end in particular the following publications are cited, the complete disclosure of which is hereby incorporated by reference:
1) G. F. Blackburn et al. xe2x80x9cElectrochemiluminescence Detection for Development of Immunoassays and DNA Probe Assays for Clinical Diagnosticsxe2x80x9d, Clin. Chem. 37 (1991), 1534-1539
2) J. K. Leland and M. J. Powell: xe2x80x9cElectrogenerated Chemilumenescence: An Oxidative-Reduction Type ECL Reaction Sequence using Triprolyl Aminexe2x80x9d, J. Electrochem. Soc., 137(1990), 3127-3131
3) J. H. Kenten et al.: xe2x80x9cImproved Electrochemiluminescent Label for DNA Probe Assays: Rapid Quantitative Assays of HIV-1 Polymerase Chain Reaction Productsxe2x80x9d, Clin. Chem. 38 (1992), 873-879
4) N. R. Hoyle: xe2x80x9cThe Application of Electrochemiluminescence to Immunoassay-based Analyte Measurementxe2x80x9d in xe2x80x9cBioluminescence and Chemilumenescensexe2x80x9d; Proceedings of the 8th International Symposium on Bioluminescence and Chemilumenescence, Cambridge, September 1994, A. K. Campbell et al. (edit.), John Wiley and Sons
5) WO 89/10551
6) WO 90/11511
As mentioned, the measurement of the ECL-light is normally carried out in a flow-through measurement cell. The cell comprises a narrow flow channel for the sample fluid, and the working electrode is disposed on one of the walls defining the flow channel. In order to be able to sequentially measure differing samples with the same measuring cell, the cell, in particular the working electrode, must be cleaned between measurements to remove the beads and other impurities deposited thereon. This cleaning process must be rapid and efficient in order to guarantee a high throughput for the analysis apparatus and good analysis precision.
Cleaning is therefore not only done physically (passage of air bubbles) and chemically (passage of a cleaning fluid containing, inter alia, a detergent). Rather also electrochemical cleaning takes place by application of a strongly oxidizing and/or reducing potential to the working electrode. The potential is normally sufficiently high that gas bubbles are formed on the surface of the working electrode. This effectively supports the cleaning process. The electrochemical equilibrium of the electrode surface is, however, so strongly perturbed that after the cleaning step a conditioning step must be carried out in which a sequence of pulses are applied to the working electrode which cover the entire working potential range of the electrode material used.
Thus, a detection cycle is carried out in the cell which includes a sequence comprising a cleaning step, a conditioning step, a capturing step and a measuring step. During the cleaning step and during the conditioning step a cleaning fluid and a conditioning fluid respectively are located in the cell. The sample fluid with the beads is introduced into the flow-through measuring cell only at the beginning of the capturing step. Heterogeneous tests comprise an additional washing step between the capturing step and the measuring step. The detection cycle is explained in more detail in references 1 through 6, primarily in WO 89/10551.
The ECL-BBA-method is distinguished, in comparison to other analysis methods which are based on the specific binding of biochemical binding partners, by simple handling, high sensitivity, a large dynamic range of measurable concentrations, an economical analysis, and good automation possibilities (by means of corresponding analysis apparatus).
In order to achieve a further increase of the analytical precision of ECL-BBA-methods of the above mentioned type an additional potential pulse having an oxidizing and/or reducing potential is introduced, in accordance with the invention, into the voltage curve of the detection cycle between the conditioning step and the capturing step to improve deposition of the microparticles, wherein the additional potential pulse is returned to a neutral (neither oxidizing nor reducing) potential before the working electrode is contacted by the sample.
The dependence of the quality of the analysis on the voltage curve applied to the working electrode during the detection cycle is discussed in WO 89/10511 (reference 5). According to this reference, a constant potential value, designated as a xe2x80x9cpreoperative potentialxe2x80x9dshould be applied at the end of the conditioning step in order to improve the reproducibility of the analysis result. This preoperative potential should remain constant until the working electrode is contacted by the sample fluid and the ECL-measurement is carried out. The preoperative potential should be either an oxidizing potential or a reducing potential in dependence on the material of the working electrode and on the electrolyte used.
In the context of the invention it has been discovered that, in contrast to the teaching of reference 5, a substantial improvement concerning the even deposition of the beads on the surface of the working electrode and thereby an improvement in the reproducibility and precision of the analysis can be achieved if the additional potential pulse is introduced into the voltage curve of the detection cycle. It is important that this pulse on the one hand attains an oxidizing or reducing potential value and on the other hand is returned to a neutral (neither oxidizing nor reducing) potential before the sample is introduced into the measuring cell and contacts the working electrode.
While the preoperative potential of WO 89/10551 is intended to influence the components of the sample pertinent to the generation of the ECL-signal, the invention achieves a substantial improvement by means of an additional electrochemical preprocessing of the electrode. The fact that this leads to an improvement in the deposition of the beads is unexpected, since it was to be assumed that the bead distribution depends on the properties of the magnetic field, whereas the electrochemical conditioning and cleaning measures serve for improvement of the signal generation.
A potential pulse as used in the invention is a transient change of the voltage applied to the working electrode during which an oxidizing or reducing potential value is reached, whereas the electric potential is in the neutral region before and after the potential pulse. The detailed shape of the potential curve may vary. It is in particular not necessary that the potential curve have a defined geometrical shape (e.g. rectangular, triangular or step function).
An oxidizing potential is an electric potential of the working electrode by which the surface thereof which is in contact with the conditioning fluid is oxidized. A potential is reducing when it effects an electrochemical reduction of the metallic surface of the working electrode in contact with the conditioning fluid. A potential by which practically no oxide or hydride layer is formed on a clean metallic surface is neutral. This state is also called the double-layer region of the corresponding metal.
No generally valid numerical values for the maximum potential of the additional pulse and for the value to which this potential must be returned can be given, since these potential values depend on the material of the working electrode, on the reference electrode to which the potential refers, and (to a lesser extent) on the composition of the conditioning liquid. Those skilled in the art can take more detailed information in this regard from published data in particular from cyclovoltamograms of the electrode material used. In any event, the values for an oxidizing, reducing, or neutral potential can be determined experimentally.